Therapeutic and diagnostic cloned mhc-unrestricted receptor specific for the muc1 tumor associated antigen

ABSTRACT

The invention provides an isolated nucleic acid encoding a receptor, other than an immunoglobulin, wherein the receptor binds to a MUC1 tumor antigen independently of an major histocompatibility complex (MHC). The invention provides a method of activating a signaling pathway and/or killing a cancer cell using a receptor that is similar to or is a T cell receptor

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application60/634,072, the disclosure of which is incorporated herein in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made in part with Government support under GrantNumbers CA56103 and predoctoral training grant DAMD17-99-1-9352 awardedby the U.S. National Institutes of Health and the U.S. Department ofDefense, respectively. The United States Government may have certainrights in this invention.

FIELD OF THE INVENTION

This invention pertains to the treatment or prevention of cancer inhumans involving transfer of (a) an isolated population of cells, or (b)cells transduced with a nucleic acid encoding a receptor, into a humanin need of treatment or prophylaxis for cancer. The present inventionalso pertains to a method of activating a signaling pathway.

BACKGROUND OF THE INVENTION

Immunotherapy of cancer involving adoptive transfer of T cells forvarious human tumor antigens has significantly improved in recent years.It has also recently become clear that immunotherapy is more potent ifboth the innate and the adaptive cellular immune responses areefficiently engaged. Timely recognition of the tumor by the cells of theinnate immune system, such as NK cells, granulocytes, and macrophages,appears to be a prerequisite for an efficient stimulation oftumor-specific adaptive immunity.

Tumor-specific antibodies have been transduced into T cells (T-bodies)endowing the transduced T cells with MHC-unrestricted tumor antigenspecificity. While antibodies can have exquisite specificity, onedisadvantage is their high affinity of binding to antigen, which couldimpair infiltration of tumors, result in irreversible binding of aneffector cell to a tumor cell, and possibly result in apoptosis ofeffector cells following their interaction with tumor cells. T cellreceptors (TCRs), however, have a much lower binding affinity.Therefore, a cell bearing a tumor-specific TCR could engage anddisengage from its target multiple times and effect its function againstmultiple tumor cells. Tumor specific T cells, however, areMHC-restricted. Accordingly, a tumor specific T cell is effective onlyfor treatment of patients having a suitable human lymphocyte antigen(HLA). Additionally, tumor cells frequently down-regulate MHC or antigenprocessing molecules, thereby avoiding T cell recognition. Despite theselimitations, adoptive immunotherapy therapies involving transfer oftumor-specific TCRs into T cells continue to be developed. Accordingly,the art is in need of an adoptive immunotherapy that can treat orprevent cancer in most or all patients. Desirably, such a therapy wouldnot be dependent on the HLA or antigen processing components of targetcancer cells. Moreover, it would be desirable if such a therapy causedlittle or no immune reaction with non-target cells.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of activating a signaling pathway in acell. The method comprises transducing the cell with at least onenucleic acid that encodes a receptor so that the receptor is expressedand displayed on the surface of the cell. The encoded receptor iscapable of binding to a MUC1 antigen without requiring the MUC1 antigento be presented in the context of a major histocompatibility complex(MHC). The receptor either interacts with a signaling molecule, orcomprises a signaling domain, such that when the receptor is contactedwith a cell having the MUC1 tumor antigen on its surface, the signalingpathway is activated. In a preferred embodiment the receptor is a T cellreceptor.

The encoded and expressed receptor can have any suitable sequence. Forexample, the receptor can have any sequence in which the affinity of thereceptor for MUC1 is determined by a first amino acid sequence and asecond amino acid sequence in which the first amino acid sequenceconsists essentially of the portion of MA Vα23 shown in FIG. 1 (SEQ IDNO:1) and the second amino acid sequence consists essentially of theportion of MA Vβ8.3 shown in FIG. 1 (SEQ ID NO:2).

The encoded receptor can also have a sequence that is at least 85%identical, at least 90% identical, at least 95% identical, or isessentially identical to the portions of MA Vα23 and MA Vβ8.3 shown inFIG. 1 (SEQ ID NOs:1 and 2, respectively).

The cell signaling can generate any useful response in the transducedcell, such as (without limitation) cytokine secretion and target cellkilling (e.g., cancer cell killing).

The invention also provides a population of cells comprises a method ofkilling a cancer cell, the method comprising isolating a population ofcells, such as T cells, that have a receptor that binds to a MUC1 tumorantigen independently of a major histocompatibility complex, andcontacting the population of cells to a cancer cell expressing the MUC1tumor antigen, thereby killing the cancer cell. The isolated populationof cells preferably does not comprise a therapeutically-effectivequantity of B cells, more preferably is substantially free of B cells,and even more preferably does not comprise B cells.

The invention also provides isolated nucleic acids encoding receptorsthat bind to the MUC1 tumor antigen even when the MUC1 tumor antigen isnot presented in the context of an MHC. The encoded receptor preferablyhas at least one amino acid sequence that is homologous with, consistsessentially of, or is identical with at least one of the amino acidsequences designated MA Vα23 or MA Vβ8.3 in FIG. 1 (SEQ ID NOs:1 and 2,respectively). The encoded receptor can be membrane bound when expressedin a cell or can be expressed in a water-soluble form.

The nucleic acid encoding the receptor can be transduced into a varietyof cells. The transduced cells can be used for therapeutic purposes(such as the treatment or prevention of cancer), diagnostic purposes(such as the identification of the MUC1 tumor antigen), or as a tool inthe study of T cell function and/or immunogene therapy.

The nucleic acids (e.g., alone or in the context of a gene deliveryvector), isolated populations of cells, and transduced cells of theinvention can be combined with sterile carriers, pharmaceuticallyacceptable excipients, or adjuvants, each of which is preferablysuitable for administration to a human.

The receptor can also be isolated or substantially purified, andcombined with a labeling agent to provide a reagent useful in thedetection of the MUC1 tumor antigen in a suitable sample.

These and other advantages of the invention, as well as additionalinventive features, will be apparent from the description of theinvention provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a comparison of the CDR2 and CDR3 regions of theMUC1-specific antibody SM3 and the MA TCR and modeling theirinteractions with MUC1. FIG. 1 a Amino acid sequence alignment of MAVα23 (SEQ ID NO:1) and MA Vβ8.3 (SEQ ID NO:2) with SM3L and SM3H (SEQ IDNOs:23 and 24, respectively). Numbering corresponds to MA TCR sequence.Residues in bold are hypothesized to be involved in the binding (FIG. 1b, and FIG. 1 c) SM3 and (FIG. 1 d, and FIG. 1 e). MA TCR computer basedmodels of the interactions with the MUC1 epitope. Contact residues areshown as stick diagrams.

FIG. 2 depicts cells transfected with scTCR recognize MUC1⁺ tumors andsynthetic MUC1 antigen. FIG. 2 a shows mammalian expression vectorencoding MA scTCR gene. The scTCR was cloned into the pEF6 vector. FIG.2 b shows cell surface expression of the scTCR in RBL cells and FIG. 2 cshows BWZ cells stably transfected with the scTCR-pEF6 vector. Cellswere stained with anti-TCR βF1 (open histogram) or with isotype control(filled histogram) antibody. FIG. 2 d shows degranulation of RBL cells(open bar) or RBL-scTCR cells (striped bar) following stimulation withplate-bound βF1 antibody or MUC1 140mer peptide. Specific-degranulationis presented as percent of maximum degranulation induced by TCRcross-linking with βF1 antibody. FIG. 2 e shows IL-2 ELISA for BWZ (openbars) or BWZscTCR (striped bar) following stimulation with plate-boundanti-TCR βF1 antibody, ionomycin+PMA (I/P), DM6 (MUC1⁻ tumor), HPAF orT3M4 (MUC⁺ tumors). IL-2 in culture supernatant was measured by ELISAand values were plotted on the y-axis as pg/ml. Cells were stimulated asindicated.

FIG. 3 shows transduction of the long-term reconstituting hematopoieticstem cell population (c-Kit⁺ Sca⁻1⁺Lin⁻ Thy1.1⁻) with scTCR-EGFP MFGretroviral vector. FIG. 3 a shows a schematic diagram of the scTCR-EGFPMFG retroviral vector. FIG. 3 b shows Bone Marrow (BM) cells transducedwith the scTCR-EGFP MFG retroviral vector were stained on day 7 inculture for hematopoietic stem cells surface markers (c-Kit and Sca-1),and for lineage markers (Lin). Cells that expressed high levels of Sca-1and c-Kit (c) and that lacked expression of Lin (d), R2 & R3 were gatedon and were plotted against EGFP (FIG. 3 e). FIG. 3 f shows mocktransduced BM cells. All cells in culture were Thy1.1⁻ (not shown).

FIG. 4 shows detection of scTCR-expressing cells at various times postreconstitution with transduced BM cells. Mice were bled at indicatedtime points and leukocytes were stained for the appropriate cell surfacemarkers plotted on the Y-axis: (FIG. 4 a) GR-1 for granulocytes, (FIG. 4b) mac-3 or F4/80 for monocyte/macrophages, (FIG. 4 c) DX5 for NK cells,(FIG. 4 d) CD3 for T cells, and (FIG. 4 e) B220 for B cells. EGFPexpression is plotted on the x-axis. Percentages of EGFP positive cellsin each lineage are indicated.

FIG. 5 shows SCID mice reconstituted with transduced BM cells cancontrol the growth of the MUC1⁺ tumor xenograft. FIG. 5 a shows control(filled triangles) or scTCR-reconstituted (open circles) mice wereinjected subcutaneously with 2×10⁶ HPAF (MUC1⁺) tumor cells. Tumor sizeis shown on the y-axis while days post-tumor challenge is plotted on thex-axis. P-Values were calculated by running t-test using Microsoft Excelsoftware. Data are presented as mean±S.E. FIG. 5 b shows H & E stainingof HPAF tumor sections from control mice (left panel) or fromscTCR-reconstituted mice (right panel). FIG. 5 c shows staining of tumorsections from scTCR-reconstituted mice for myeloperoxidase (neutrophilsmarker), F4/80 (monocytes/macrophage marker), or Granzyme B (NK cellsmarker). Images were taken under 20× magnification. Images in the lowerright squares were taken under 100× magnification.

FIG. 6 shows expression of scTCR on immune cells has no deleteriouseffects on MUC1⁺ normal tissues. FIG. 6A shows C57BL/6 (wild type) andMUC1 Tg. mice were reconstituted with BM cells transduced with thescTCR-EGFP MFG retroviral vector. Untreated mice served as controls.Spleen, lung, and pancreas were harvested 6 weeks postreconstitution andmicroscopically examined for infiltration with EGFP⁺ cells (FIG. 6 a) orstained with H&E (FIG. 6 b) and examined for tissue destruction.

FIG. 7 shows scTCR-reconstituted MUC1 Tg. mice rejected MUC1+ tumorchallenge. MUC1 Tg. mice were reconstituted with BM cells transducedwith scTCR (filled triangle) or with control supernatant (filled square)and challenged 6 weeks later with MUC1 tumor (RMA) or with RMA cellstransfected with MUC1 (RMA-MUC1). Data are presented as Mean±S.E. Thenumber of mice at each time point ranged from 5-10.

FIG. 8 shows RT-PCR analyses of scTCR expression in transduced BM cellsand in splenocytes from reconstituted SCID mice. FIG. 8 a showsexpression of scTCR mRNA in transduced BM cells 72 hourspost-transduction. FIG. 8 b shows expression of scTCR mRNA was insplenocytes 60 days post-reconstitution. M is 1 Kb DNA molecular weightmarker. β-actin is the RT-PCR control.

FIG. 9 shows long-term expression of scTCR in mice reconstituted withscTCR-transduced BM cells. Control (FIG. 9 a) or scTCR-reconstitutedmice (FIG. 9 b) were bled 7 months post-reconstitution and were stainedfor GR-1. Cells were gated on GR-1+ and were plotted against EGFP.

FIG. 10 shows expression of the TCR αβ from MA CTL clone on the surfaceof a TCR-deficient Jurkat line (JRT3-T3.5). The TCR α-IRES-β cassettewas cloned into the pEF4 expression vector (FIG. 10A), UntransfectedJRT3-T3.5 (FIG. 10B), JRT3-T3.5 cells transfected with the TCR β chain(FIG. 10C), or JRT3-T3.5 cells transfected with MA TCR α-IRES-β pEF4(FIG. 10D), were stained with anti-CD3ε (open histogram) or with isotypecontrol (filled histogram) antibody. IRES means internal ribosomal entrysite, and Zeocin is an antibiotic resistance gene.

FIG. 11 shows the construction and expression of MUC1-specific αζ and βζchimeric T cell receptors. Expression vectors for TCR αζ (FIG. 11A), βζ(FIG. 11B), and αζ/βζ (FIG. 11C). Untransfected 293H cells (FIG. 11D),293H cells co-transfected with the TCR αζ and TCR βζ (FIG. 11E) or cellstransfected with the αζ-IRES-αζ pLNCX2 (FIG. 11F) were stained forsurface expression of the TCR using anti-TCR antibody βF1 (openhistogram) or isotype control antibody (filled histogram).

FIG. 12 shows vector construction and expression of MUC1-specific singlechain T cell receptors (scTCRs). 293H cells (FIG. 12A) were transfectedwith the scTCR (FIG. 12B), scTCR-CD4TM-hζ (FIG. 12C), orscTCR-CD4TM-AGD-hζ (FIG. 12D) mammalian expression vectors. Cells werestained with anti-TCR βF1 (open histogram) or isotype control (filledhistogram) antibody. FIG. 12E shows quantitative comparison of TCRexpression on 293H cells transfected with different scTCR constructs.p<0.05.

FIG. 13 shows expression of functional scTCR on the surface of T cellsand non-T immune cells. (FIG. 13A) Rat Basophilic Leukemia (RBL) ormouse T cell tumor BWZ cells were transfected with the scTCR-pEF6 andstained for surface expression with anti-TCR βF1 antibody (filled) orwith isotype control (open) histogram. FIG. 13B shows IL-2 secretionfrom BWZ cells (open bars) or BWZ-scTCR (filled bars) followingstimulation with SEE superantigen or with anti-TCR βF1 antibody.

FIG. 14 shows expression and purification of soluble scTCR (sscTCR)following surface biotin labeling and immunoprecipitation. FIG. 14Ashows scTCR expression vector encoding a thrombin cleavage site, T. FIG.14B shows RBL cells transfected with the scTCR were stained withanti-TCR βF1 antibody (open histograms) before (right) or after (left)treatment with thrombin. The filled histogram shows staining withisotype control antibody. FIG. 14C shows immunoprecipitation of thescTCR from RBL (lanes 1 and 2) or RBL cells transfected with the scTCR(Lanes 2 and 4) before (lanes 1 and 2) or after (lanes 3 and 4)treatment with thrombin. Lane 6-8 are SA-HRP blotting of fraction elutedwith 150 mM Glycine, PH 2.2, 100 mM Glycine pH 2.2, or diethyl amine(DEA) pH 11.2, respectively. Lane 5 is IP from control lysate.

FIG. 15 shows expression and purification of soluble scTCR using amammalian expression system. FIG. 15 a shows the single chain fractionvariable (scFV) domain was cloned and fused to a C-terminus HA and c-mycepitope tags. FIG. 15 b shows secreted scTCR (sscTCR) that was clonedand fused to a C-terminus Flag and 6-His epitope tags. FIGS. 15 c and 15d show the sscTCR as described in FIG. 15 b, except fused to the leadersequence from GM-CSF (FIG. 15 c) or from Ig-κ light chain (FIG. 15 d).FIG. 15 e shows a western blot of the culture supernatants from 293Hcells transiently transfected with constructs a-d (a′-d′),immunoprecipitated with appropriate anti-tag antibody and blotted withanti-c-myc antibody (a′) or with anti FLAG-M2 antibody (b′-d′). “Ctr.”is supernatant from untransfected cells. (f) Comassiee blue staining offractions from culture supernatant b purified using nickel column. Lane1 is culture supernatant before purification, 2 is flow through, 3 iswash, and 4-6 are different eluted fractions. FIG. 15 g shows a westernblot of panel f using anti-Flag M2 antibody.

DETAILED DESCRIPTION OF THE INVENTION

The MA TCR referenced in FIG. 1 is a T cell receptor (TCR) that binds tothe MUC1 tumor antigen. The MA TCR recognizes an epitope located in eachof the 20 amino acid long tandem repeats in the extracellular domain ofMUC1. Each molecule of MUC1 can have more than 100 repeats. The aminoacid sequence critical for recognition by the TCR is believed to be afive amino acid residue sequence (PDTRP SEQ ID NO: 3) which has beencalled the immunodominant knob of the MUC1 protein. A large number ofthese tandemly repeated and structurally stable PDTRP (SEQ ID NO: 3)bearing knobs on a single MUC1 molecule, as well as on neighboring MUC1molecules on the surface of a tumor cell, can engage multiple TCRs, andsignal a T cell to effect its function. While not desiring to be boundby any particular theory, it is believed that the MA TCR can effectivelybind with the MUC1 tumor antigen without requiring antigen processingand presentation in an MHC, which is required by most T cell receptors.While the MA TCR referenced in FIG. 1 is a preferred TCR that is capableof recognizing the MUC1 tumor antigen independently of presentation byan MHC, functionally similar TCRs can be raised against the MUC1 tumorantigen or any other disease associated antigen with structurallystable, repeated amino acid epitope similar to MUC1. The antigen bindingdomains of these TCRs readily can be incorporated into a variety ofreceptor constructs such that a cell expressing the receptor will haveat least one signaling pathway activated when contacted to a cell havingthe MUC1 or other structurally stable, repeated amino acid epitope onits surface.

Accordingly, the invention provides a method of activating a signalingpathway in a cell. The method comprises transducing the cell with atleast one nucleic acid encoding a receptor that binds to the MUC1 tumorantigen independently of presentation of the MUC1 tumor antigen in thecontext of an MHC. The signaling pathway is activated when the receptoris expressed and displayed on the surface of the transduced cell, andthe transduced cell is contacted to a cell having the MUC1 tumor antigenon its surface. Of course, the transduced cell can be a T cell, but asis further described below, any suitable cell or collection of cells canbe transduced.

The invention also provides an isolated nucleic acid encoding thereceptor. Nucleic acids suitable in the context of the invention includenatural and synthetic nucleic acids. Natural nucleic acids can be DNA orRNA irrespective of whether they are isolated directly from a cell,e.g., an MA T cell clone, synthesized by chemical or other means, orcarried in a vector. Artificial nucleic acids can include regions ofnatural polynucleotides and can further comprise 2′ methyl nucleicacids, 2′ methoxy nucleic acids, phosphorothiorated nucleic acids,nucleotides comprising synthetic or modified bases (e.g., inosine),peptidyl nucleic acids (PNAs) and other synthetic nucleic acids known inthe art, so long as the nucleic acid can be transcribed, if necessary,and translated into a receptor. The receptor encoded by the isolatednucleic acid is preferably not an antibody or immunoglobulin. Similarly,the receptor preferably does not have a constant region of any antibody.

The sequence of the receptor does not need to be identical to that ofthe MA TCR. In fact, amino acid substitutions can be made or allowed inboth the affinity determining regions of the MA TCR and in the frameworkregions of the TCR. Additionally, domains of other proteins can beincorporated into the receptor. Accordingly, the invention also providesa method of activating a signaling pathway comprising transducing a cellwith a nucleic acid encoding a receptor having affinity for the MUC1tumor antigen, wherein the affinity of the receptor for the MUC1 tumorantigen is determined by a first amino acid sequence consistingessentially of the portions of MA Vα23 and MA Vβ8.3 shown in FIG. 1 (SEQID NOs:1 and 2, respectively).

Additionally, the invention provides a method of activating a signalingpathway or of killing a cancer cell wherein the transduced cell, orpopulation of cells, comprises a receptor having at least one amino acidsequence that has at least 85%, optionally at least 90%, or optionallyat least 95% identity with the portion of MA Vα23 shown in FIG. 1 (SEQID NO:1). The receptor can also have a sequence that is identical withthe portion of MA Vα23 shown in FIG. 1 (SEQ ID NO:1). The receptorpreferably also has at least one amino acid sequence that has at least85%, optionally at least 90%, or optionally at least 95% identity withthe portion of MA Vβ8.3 shown in FIG. 1 (SEQ ID NO:2), or which isidentical with the portion of MA Vβ8.3 shown in FIG. 1 (SEQ ID NO:2).More preferably, the receptor comprises both amino acid sequences.

As used herein, a sequence is 85% identical to another sequence if, in awindow comprising the number of amino acids present in the namedsequence, at least 85% of the amino acids, allowing gaps or insertionswithin the window (but not allowing the number of amino acids in thewindow to exceed the number of amino acid residues in the test sequence)are the same as those in the other sequence. For example, the portion ofMA Vα23 shown in FIG. 1 has 110 amino acid residues. Accordingly, asequence is 85% identical with the portion of MA Vα23 if at least 94residues in any 110 consecutive residues of one sequence can be exactlyaligned with 110 consecutive residues of the other sequence.

The receptor can also be any receptor having an amino acid sequencecomprising the complementarity determining regions (CDRs) of MA Vα23 orMA Vβ8.3 or both.

The invention also provides a method of killing a cancer cell. Thecancer cell can be present in a culture of cells in vitro or found in ananimal's (e.g., a human's) body. The method of killing the cancer cellinvolves isolating a population of cells comprising a receptor thatbinds to a MUC1 tumor antigen independently of an MHC and contacting aportion of the cells from the isolated population of cells to a cancercell. The population of cells can be created by transduction of one ormore cells of an animal with a nucleic acid of the invention. In anotheralternative, an animal can be immunized with the MUC1 tumor antigen toraise lymphocytes comprising a receptor that mediates the death of cellsexpressing the MUC1 tumor antigen. While not desiring to be bound by anyparticular theory, it is believed that the highly repetitive nature ofthe MUC1 epitope enables the routine generation of T cells expressingreceptors for the MUC1 antigen that are MHC unrestricted. Of course, thepopulation of cells can comprise T cells or consist essentially of Tcells. The population of cells also can be isolated or manipulated insuch a way that the population does not comprise any cells other than Tcells that comprise a receptor that binds to a MUC1 tumor antigenindependently of an MHC. In one embodiment, for example, the populationof cells does not comprise B cells having a B cell receptor (BCR) thatis specific for the MUC1 tumor antigen.

The receptor can have any suitable affinity for the MUC1 tumor antigen.For example, the affinity of the receptor can be higher than theaffinity of the MA TCR for the MUC1 tumor antigen. Receptors havinglower affinity for the MUC1 tumor antigen than the MA TCR are alsouseful in the context of the present invention. However, the receptorpreferably has about the same affinity of the MUC1 tumor antigen as theMA TCR because this level of affinity is high enough to efficientlyinvoke effector functions of T cells, while simultaneously allowingefficient tumor infiltration and avoiding apoptosis of the cell on whichit is displayed. While receptors having other affinities for the MUC1tumor antigen are useful, receptors having a K_(d) of between 0.2 μM and200 μM, are among the preferred embodiments. The affinity can bemeasured with a variety of conventional techniques. Preferably, however,the receptor is converted into a soluble form (if necessary) to measureits affinity. The skilled artisan will appreciate that conventionaltechniques employing a Biacore® device are particularly well suited tothe measurement of receptor affinities for the MUC1 tumor antigen(Molloy et al., Molecular Immunology, 35: 73-81 (1998)).

The transduced cell, or a cell of the isolated population of cells,desirably has a suitable avidity for a cancer cell expressing the MUC1tumor antigen. For example, the avidity between the transduced cell, orcell of the isolated population, can be between 1×10⁻⁵ M and 1×10⁻¹² M.

The receptor can be encoded by 1, 2, or more nucleic acids. Any receptor(or nucleic acid encoding a receptor) having the antigen determiningregions of the MA TCR is suitable in the context of the invention,including (but not limited to) a receptor encoded by a single nucleicacid, such as, but not limited to, a single-chain receptor in which theregions of the receptor having homology to the α-chain and the β-chainof the MA TCR are encoded as a single polypeptide. The receptor can alsobe an scFv. In other embodiments, the receptor can be encoded as atleast two subunits. The receptor desirably has amino acid sequencesderived from the Vα, a Jα, Vβ, Dβ, and Jβ mature gene segments of the MAreceptor. Additionally, the receptor preferably does not have light andheavy chains or an antibody, nor truncated polypeptides derived fromlight and heavy chains or the nucleic acids encoding the same.

In a preferred embodiment the receptor comprises at least onepolypeptide that is fused to, or connected with, a portion of thezeta-chain of CD3. Many suitable configurations exist. In a preferredembodiment, the transmembrane and cytoplasmic domains of the zeta-chainof a CD3 molecule are fused to the α-constant domain of a TCR or, morepreferably, a β-constant domain of a TCR. While not desiring to be boundby any particular theory, it is believed that the fusion of a portion ofthe CD3 zeta-chain to the remainder of the receptor facilitates surfaceexpression of the receptor on a transduced cell. In a more preferredembodiment, the constant domain of the TCR and the portion of the CD3zeta-chain are separated by an amino acid linker (also known as an aminoacid spacer) encoded by a nucleic acid encoding the entire receptor or aportion of the receptor. The linker can be any suitable sequence, but ispreferably selected to have a flexible structure. Any number of aminoacid residues can be included in the linker but typically the linkerwill comprise at least one, more typically at least three, yet moretypically at least about eight, and commonly at least about 12 aminoacid residues. Additionally, the linker will typically not comprise morethan about 30 amino acid residues, more commonly not more than about 22amino acid residues, and commonly not more than about 18 amino acidresidues. A linker having about 15 amino acid residues is among the morepreferred embodiments. Additionally, the linkers primary function is toprovide a flexible linkage between two portions of the receptor.Accordingly, the linker can optionally not have one or more functionsselected from the group consisting of an immunological function, amembrane anchoring function, a membrane spanning function, adimerization function, a signaling function, and an intracellulartrafficking function. In contrast, however, the receptor can have aminoacid sequences in additional to the linker which provide at least one ofthese functions. The linker preferably allows a high level of receptorexpression when the receptor is expressed in a T cell or other suitablecell in comparison to an otherwise identical receptor that lacks thelinker.

In another embodiment, the receptor comprises the transmembrane regionfrom a CD4 molecule.

In embodiments comprising portions of a CD3 or a CD4 molecule theportions of these molecules can contain substantial variations fromtheir natural sequences. For example, the portions of the CD3 or CD4molecules can have at least about 65% identity, at least about 80%identity, or be essentially identical to a consensus sequence (alsoknown as “wild type”) human CD3 or CD4 molecule.

The nucleic acid encoding the receptor can be carried by any suitablevector. The vector is preferably a gene transfer vector (also known as agene delivery vector). Suitable gene transfer vectors include both viraland non-viral vectors. Suitable viral vectors include, but are notlimited to, retroviral vectors. Suitable viral vectors include, but arenot limited to adenoviral vectors, adeno-associated viral vectors, andherpes viral vectors. Lentiviral vectors, particularly those having theability to efficiently transduce quiescent cells are among the morepreferred viral vectors. Other preferred vectors in the context of thepresent invention include liposomal vectors and MFG vectors.

The nucleic acids encoding a receptor having affinity for the MUC1 tumorantigen of the invention can be transduced into a stem cell, preferablya bone marrow stem cell. Advantageously, this enables the generation ofa wide variety of cell types expressing the receptor, each of which hasa high potential for propagation. Similarly, the cells can be removedfrom an animal and transduced in vitro. When the cells are transduced invitro they need not be, but optionally can be, expanded (i.e.,propagated) in vitro prior to their transfer into a host animal such asa human. Any suitable technique can be used to propagate the cells invitro. Suitable methods include, but are not limited to culturing thecells with cytokines, stimulatory molecules (e.g., stimulatoryantibodies), live, attenuated, or killed cells having the MUC1 tumorantigen on their surface. Preferably, the cells are autologous, whichmeans that they are transferred into the host from which they wereobtained. Transduced autologous cells are believed to be less likely tobe rejected by the host animal into which they are transduced.

Conveniently, the nucleic acid(s) encoding the receptor can betransduced into the cells in vivo. While not desiring to be bound by anyparticular theory, it is believed that the transduced cells will findcells expressing the MUC1 tumor antigen on their surface, if present,and be stimulated to expand to reach therapeutic levels when atherapeutic quantity of cells is not transduced directly.

The cell can be transduced in a mixed population of cells, or can bepartially or substantially purified prior to transduction with thenucleic acid encoding the receptor. A preferred cell type fortransduction is the fibroblast. Fibroblasts are capable of rapidexpansion and transduced fibroblasts treat and prevent cancer inpatients having cancer cells that expresses the MUC1 tumor antigen.

The transduced cells can also be of the hematopoietic lineage.Hematopoietic cells that can be usefully transduced in the context ofthe present invention include T cells, B cells, NK cells, macrophages,granulocytes, and dendritic cells.

In a preferred embodiment, the cell to be transduced is removed from ananimal (e.g., a human or other animal), the animal is treated forcancer, e.g., with chemotherapy or radiation or both. The chemotherapyand/or radiation optionally can be potent enough to decimate or destroythe patient's lymphocyte population. Then, the transduced cells arereturned to the patient. Before, during, or after the transduced cellsare transferred to the patient or other animal, the patient or otheranimal can be treated with cytokines that expand the population oflymphocytes (e.g., IL-2). Additionally, other therapeutic agents such asantiemetics, erythropoietin or other red blood cell boosters, hormones,and antibodies can also be administered to the patient or animal before,during, or after the transfer of the transduced cells to the patient oranimal. Similarly, the animal can be treated with a vaccine, whichvaccine is preferably a vaccine promoting a reaction against a tumor orcancer cell, and more preferably is a MUC1 vaccine. The MUC1 vaccinestimulates cells reactive with MUC1 to persist, or preferably, topropagate. Suitable MUC1 vaccines can comprise at least one PDTRP (SEQID NO: 3) sequence, which pentapeptide optionally can be found in thecontext of from about 5 to about 20 amino acids having identity with anaturally-occurring MUC-1 polypeptide known in the art. Such a vaccinepreferably would comprise a multiplicity of PDTRP (SEQ ID NO: 3) aminoacid sequences so that a MUC1 reactive cell would be presented with anarray of reactive epitopes. For example, the vaccine can be configuredto present reactive cells with 2, 3, 4, 5, 6-10, 11-20, or more PDTRP(SEQ ID NO: 3) epitopes in an array or complex, preferably on thesurface of a cell. The vaccine can further comprise an excipient,sterile carrier or adjuvant.

In embodiments in which contact of the receptor with the cell having aMUC1 tumor antigen on its surface activates a signaling pathway, thesignaling pathway can be of any suitable type. For example, thesignaling pathway can cause the secretion of a cytokine or induce acytotoxic response leading to the death of the cell displaying the MUC1tumor antigen. Similarly, activation of the signaling pathway can leadto the secretion of a biomolecule, such as a endocrine, paracrine,autocrine, whether steroidal, peptidyl, or other, an enzyme, acarbohydrate, a proinflammatory or anti-inflammatory molecule, or a drugagent. In a preferred embodiment, the biomolecule causes the death of acancer cell or attenuates the symptoms of cancer.

The receptor encoded by any nucleic acid of the invention can also besubstantially purified or isolated from the cell expressing it,irrespective of whether the receptor is water-soluble or has a highaffinity for cellular membranes (or artificial membranes such asmicelles).

The inventive receptor can also be complexed with a labeling agent. Suchreceptor-labeling agents complexes are useful for the visualization ordetection of cells expressing a MUC1 tumor antigen. A labeling agent canbe directly detectable or indirectly detectable. For example, directlydetectable labeling agents include (without limitation) a protein suchas green fluorescent protein (i.e., GFP), a radioactive atom, or a goldmicroparticle can be attached to the receptor. Each of these moietiescan be directly detected with suitable, conventional methods.Alternatively, the label can be an indirect label such as (withoutlimitation) an enzyme (e.g., horse radish peroxidase), an antibodyhapten (e.g., the well-known FLAG epitope, biotin, avidin, orstreptavidin, or the like. The complex can be covalently ornon-covalently bound as long as the complex remains associated duringany detection steps. Additionally, the complex can be, but need not be,purified or substantially isolated from a cell expressing the receptor.

In view of the foregoing, the invention also provides an isolated cellthat is optionally transduced with a nucleic acid of the invention, andencodes a receptor of the invention.

Any of the nucleic acids, cells, or population of cells of the inventioncan be combined with a sterile carrier, pharmaceutically acceptableexcipient, or adjuvant, each of which is preferably suitable foradministration to a mammal, and in particular a human. The compositioncan comprise a buffer, and the buffer or composition is preferablysubstantially isotonic with human blood (e.g., is isotonic with 0.7 M to1.1 M NaCl₂).

Suitable methods of administering the nucleic acids of the invention toa mammal for purposes of gene therapy are known (see, e.g., Rosenfeld etal., Science, 252, 431-434 (1991); Jaffe et al., Clin. Res., 39, 302A(1991); Rosenfeld et al., Clin. Res., 39, 311A (1991); Berkner,BioTechniques, 6, 616-629 (1988); Crystal et al., Human Gene Ther., 6,643-666 (1995); Crystal et al., Human Gene Ther., 6, 667-703 (1995)).Innate and adaptive cells can be found in most locations in themammalian body. Accordingly, any suitable route of administration can beused. Intravenous administration of cells is preferred when the mammalis human. A particular route can provide a more immediate and moreeffective reaction than another route. Pharmaceutically acceptableexcipients also are well-known to those who are skilled in the art, andare readily available. The choice of excipient will be determined inpart by the particular method used to administer the recombinant vector.Accordingly, there is a wide variety of suitable formulations for use inthe context of the invention.

Moreover, to optimize the ability of vectors, and particularly viralvectors, to enter a cell by the method of the invention, preferably themethod is carried out in the absence of neutralizing antibodies directedagainst the particular vector being introduced intracellularly, whichcould impede transduction of target cells. The ordinarily skilledartisan can routinely test for the presence of such neutralizingantibodies. Techniques are also known in the art to prevent the presenceof neutralizing antibodies from impeding effective protein production(see, e.g., International Patent Application WO 96/12406).

The following methods, formulations, and excipients for administeringthe inventive nucleic acids, vectors, and cells are merely exemplary andare in no way limiting.

Formulations suitable for oral administration of the nucleic acids andvectors can consist of (a) liquid solutions, such as an effective amountof the compound dissolved in diluents, such as water, saline, or orangejuice; (b) suspensions in an appropriate liquid; and (c) suitableemulsions. Tablet forms can include one or more of lactose, mannitol,corn starch, potato starch, microcrystalline cellulose, acacia, gelatin,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, stearic acid, and other excipients, colorants, diluents,buffering agents, moistening agents, preservatives, flavoring agents,and pharmacologically compatible excipients.

Preferred formulations include aqueous and non-aqueous, isotonic sterileinjection solutions, which can contain anti-oxidants, buffers,bacteriostats, and solutes that render the formulation isotonic withblood, and aqueous and non-aqueous sterile suspensions that can includesuspending agents, solubilizers, thickening agents, stabilizers, andpreservatives. The inventive nucleic acids and vectors can be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

The nucleic acids, vectors and cells of the invention can be formulatedin unit-dose or multi-dose sealed containers, such as ampules and vials,and can be stored frozen. These nucleic acids, vectors and cells of theinvention can be stored in light-resistant packaging, employing forexample, colored glass vials or cardboard boxes. Similarly, instructionsfor use of the compositions, which preferably comply with theregulations of the U.S. Food and Drug Administration, and morepreferably also with its European and Japanese equivalent agencies, canbe included with these compositions. These nucleic acids, vectors andcells of the invention are preferably also free from non-recombinantmicrobes (including without limitation fungi and mycobacteria) andnon-recombinant viruses. Preferably, the instructions suggest the use acertain quantity of one of these compositions (or range of quantities),or suggest administration of the composition to a mammal for research ortherapy via a particular route of administration.

Additionally, a cell, and more preferably, a nucleic acid or vector ofthe invention can be made into suppositories by mixing with a variety ofbases such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration can be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulas containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

The dose administered to an animal, particularly a human, in the contextof the invention will vary with the inventive embodiment, thecomposition employed, the method of administration, and the particularsite and organism being treated. However, the dose should be sufficientto provide a therapeutic response.

Any suitable number of transduced cells, or isolated cells, can beadministered to a mammal. While a single cell of the innate or adaptiveimmune system is capable of expanding and providing a benefit, when thetransduced or isolated cell is not a stem cell, it is preferable toadminister at least 10³, more preferably at least 10⁵, even morepreferably at least 10⁸ and optionally 10¹² or more transduced T cells.One preferred embodiment of the invention comprises administration offrom about 10⁸ to about 10¹² transduced cells to a human. There is notheoretical upper limit on the number of transduced T cells that can beadministered to a mammal or the number of times that T cells can beadministered to a mammal. The ordinarily skilled artisan willappreciate, however, that the excessive quantities of administeredtransduced or isolated cells (e.g., in some embodiments more than 10¹⁸transduced cells) can exceed the mammal's ability to support them, leadto undesirable clinical sequelae, and unnecessarily increase costs.Similarly, excessive administrations of therapeutic compositions tomammals can lead to undesirable effects such as allergic responses andinfection, and so are preferably avoided.

A composition comprising transduced cells can be prepared so that itdoes not contain living cells other than blood cells and lymphocytes.That is, the composition can be sterile except for the transduced orisolated cells. Such compositions can be readily prepared by positiveand negative selection of the desired cells from a population of cellstransduced with the inventive nucleic acids or vectors. Suitablepositive selection techniques include bioaffinity separations, which arewell known in the art. For example, an antibody specific for a cellsurface antigen of a desired cell can be linked to a magnetic bead,incubated with the transduced population, separated therefrom andoptionally washed. Similarly, undesired cells can be eliminated from thecomposition by any suitable technique. Suitable negative selectiontechniques include immunomagnetic removal of undesired cells, and theuse of antibiotics to destroy microbes. Moreover, leukophoresis, otherfiltration techniques, sterile technique, differential centrifugation,and other conventional methods can be used to produce a compositionsuitable for administration to a human.

In embodiments in which the mammal is subjected to lymphodepletion andcytokine or growth factor stimulation, any suitable regimen can be used.Dudley et al., Science, 298: 850-854 (2002), Rosenberg et al., J. Natl.Cancer Inst., 86: 1159-1166 (1994) and Dudley et al., J. Immunother.,25: 243-251 (2002), as well as other references described in thesereferences, discuss one suitable lymphodepletion and IL-2 stimulationregimen. These references suggest, for example, treatment of a humanwith cyclophosphamide (about 60 mg/kg) and fludarabine (about 25 mg/m²)and high-doses of IL-2 (i.e., about 720,000 IU/kg). Administration ofIL-2 is preferably repeated multiple times and more preferably repeated3 to 15 times, and is preferably administered 1-5 times daily, whichnumbers can be selected and adjusted within the discretion of theskilled medical artisan.

EXAMPLE 1

This example shows that a TCR with unique MHC-unrestricted antigenbinding properties for the tumor antigen MUC1 binds its epitope on theMUC1 protein without the requirement of processing and presentation.This example also shows that a single chain Vα/Vβ/Cβ (scTCR) fused to aCD3 zeta-chain (ζ-chain) facilitates expression on the surface of cellsof the innate immune system (e.g., granulocytes, macrophages, naturalkiller cells (NK cells)) as well as the adaptive immune system (e.g., Tcells and B cells). This example additionally shows that cells of theinnate immune system reject a tumor when provided with a tumor-antigenspecific TCR. Also, this example shows that bone marrow cells transducedwith a gene transfer vector encoding a T cell-related receptor specificfor the MUC1 tumor antigen can improve the health of a mammal havingcancer in which the cancer expresses a MUC1 tumor antigen. This examplealso shows that expression of the MUC1 tumor antigen specific receptorof the invention on large percentages of cells does not result ininfiltration or destruction of tissues expressing normal MUC1.Additionally, this example shows that long-term expression of theinventive receptor can be achieved, particularly by transducing asuitable stem cell.

The following sections describe the Materials and Methods used in thisexample.

Computer Modeling. The amino acid sequence of the SM3 antibody shown inFIG. 1 was provided by Dr. J. Taylor-Papadimitriou, of the ImperialCancer Research Fund. The Protein Data Bank at the ResearchCollaboratory for Structural Bioinformatics (www.rcsb.org/pdb) wassearched for best-fit sequence alignments with the binding domain of theSM3 antibody using BLAST. Coordinates of a crystallized human α/β TCRheterodimer were provided by Dr. Ian Wilson, of the Scripps Clinic.Modeling studies were conducted on a Silicon Graphics Indigoworkstation. Homolog templates were mutated and initial models wereconstructed using the program O (Jones, et al., Acta Crystallogr A 47(Pt 2):110-119 (1991)). The programs LEaP (Schafmeister, et al. “LeAP”,University of California, San Francisco (1995)) and AMBER/Interface wereused to import protein database (PDB) files created by O into AMBER,which was used for global energy minimizations and molecular dynamics ofselected loops and modeling of ligand binding (Pearlman, et al. ComputerPhysics Communications 91: 1-41 (1995)). The previously solved structureof the MUC1 epitope, PDTRP (SEQ ID NO: 3), and its 4 flanking aminoacids (Fontenot et al. J Biomol Struct Dyn 13: 245-260 (1995)) wasinitially positioned in the antigen-binding region by rigid body dockingusing O. This positioning was followed by energy minimization andmolecular dynamics in AMBER to allow the MUC1 epitope to position itselfwithin the antigen-binding cleft. Figures were constructed usingMolscript and Raster3D (Kraulis, et al., Journal of AppliedCrystallography 24: 946-950 (1991), Merritt, et al. Methods inEnzymology 277: 505-524 (1997)).

Construction of scTCR and Expression in RBL and BWZ Cells. The RNAencoding the Vα23.1Jα14.3 of the MA TCR was cloned from the MA CTL cloneby RT-PCR and ligated in frame to the TCR Vβ8.3DPβJβ1.2 region using a15 amino acid (aa) flexible linker encoding sequence GGG GSG GGG SGG GGS(SEQ ID NO: 4). The cloned sequence encoding theVα23.1Jα14.3-linker-Vβ8.3DβJβ1.2 polypeptide was then cloned into avector encoding the human TCR fragment Cβ2, followed by a polypeptidelinker, GDLVPRGSSRLD (SEQ ID NO: 5) and the murine CD3 ζ-chain (obtainedfrom Dr. A. J. McMichael, Weatherall Institute of Molecular Medicine,John Radcliffe Hospital, Oxford, UK) (Callan, et al., Eur J Immunol 25:1529-1534 (1995)). The last cysteine in the Cβ2 region was mutated toalanine using site directed mutagenesis to prevent dimerization of thescTCR. The construct was then cloned into the pEF6 mammalian expressionvector (Invitrogen, Carlsbad, Calif.). RBL cells were obtained from Dr.Richard Klausner (while at NCI), and BWZ murine T cells were obtainedfrom Dr. Nilabh Shastri (University of California, Berkeley). RBL cellswere grown in cDMEM-10 medium (Mediatech Inc., Herndon, Va.; DMEM+10%FBS, 2 mM L-glutamine, 100 U/ml Penicillin, 100 U/ml streptomycin, 10 μM2-ME, 1× non-essential amino acids, and 1× sodium pyruvate), while BWZcells were grown in cRPMI-10. RBL and BWZ cells were transfected byelectroporation using Bio-Rad Gene Pulser II (Bio-Rad Laboratories,Hercules, Calif.) at 960 μF and 200 V settings.

Basophil Degranulation Assay. RBL cells or RBL-scTCR were incubated with³H-serotonin (New England Nuclear Corp., Boston, Mass.) for 24 hours.After washing, cells were transferred to plates coated with βF1 antibodyor with MUC1 140mer synthetic peptide (seven repeats of the sequencePDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 6)). Plates were centrifuged brieflyand incubated for 30 minutes at 37° C. Ice-cold PBS (Sigma) was addedand the supernatant was harvested after additional centrifugation.Radioactivity was measured using a Wallac 1205 betaplate liquidscintillation counter (Gaithersburg, Md.).

IL-2 ELISA. BWZ or BWZ-scTCR cells were plated in U-bottom 96 wellplates at 1×10⁵/200 μl of cRPMI-10 medium, and 2×10⁴ tumor cells(irradiated 6000 rad) were added as stimulators. Thirty six hours later,the amount of mIL-2 released in the medium was measured using a mouseIL-2 OptEIA kit (BD Pharmingen), according to the manufacturer'srecommendations.

Construction of scTCR-EGFP MFG Retroviral Vector and Production of ViralSupernatant. The MFG retroviral vector was obtained from Dr. PaulRobbins (University of Pittsburgh, Pittsburgh, Pa.). The IRES-EGFPcassette was cloned by PCR from the pIRES2-EGFP vector (ClontechLaboratories, Palo Alto, Calif.) into the MFG retroviral vectordownstream of the scTCR gene. The GP+E-86 ecotropic retroviral packagingcells (American Type Culture Collection, Manassas, Va.) were transfectedwith the scTCR-EGFP MFG vector and cultured for 5 days, followed bysorting the EGFP^(high) population. Sorted cells were then cultured inDMEM-15 (DMEM+15% FBS, 2 mM L-glutamine, 100 U/ml Penicillin, 100 U/mlstreptomycin) and thirty six hours later, retroviral supernatant washarvested and frozen at −80° C. until use.

Retroviral Transduction of BM Cells. All experiments in animals wereperformed under an approved protocol No. 0304530A-1 of the University ofPittsburgh IACUC. Six to eight week old SCID or Balb/c mice (JacksonLaboratory, Bar Harbor, Me.) were injected intraperitoneally with 150mg/kg 5-FU (Invivogen, San Diego, Calif.). Five days later, mice weresacrificed and BM cells were isolated and pre-stimulated for 72 hours inthe presence of 50 ng/ml rSCF, 10 ng/ml rmIL-3 and 10 ng/ml rmIL-6(PeproTech Inc., Rocky Hill, N.J.) in DMEM-15. BM cells were resuspendedin retroviral supernatant supplemented with 50 ng/ml rSCF, 10 ng/mlrmIL-3, 10 ng/ml rmIL-6 and polybrene at 8 μg/ml. In all BM transductionexperiments, cells were plated in 24-well plates pre-coated withrecombinant fibronectin CH-296 fragment (Takara Bio Inc, Madison, Wis.).Cells were then centrifuged for 30 minutes at RT at 800×g in a SorvallT6000B centrifuge and put back in culture at 37° C. Transduction wasrepeated every 12 hours for a total of 4 cycles.

Reconstitution of Irradiated Mice with Transduced BM Cells and FlowCytometric Analyses. Transduced BM cells were resuspended in PBS at1×10⁷/ml, and 200 μl of cell suspension was injected via the tail veininto irradiated recipient mice. SCID mice received a single dose of 350rad while Balb/c mice were given a split dose of 900 rad total (50/50) 3hours apart. Reconstituted mice were maintained in a germ-freeenvironment and were put on acidified water (pH 2.5) for 3 weekspost-reconstitution. At 3, 6, and 11 weeks post-reconstitution, 200 μlof blood was collected via tail artery and cells were stained with theappropriate anti-surface marker antibody, PE-conjugated anti-CD3, -B220,-GR-1, -Mac-3 and -DX5 and APC-conjugated anti-F4/80 (eBiosciences, SanDiego, Calif.), and analyzed on the Becton-Dickinson FACSCalibur (BDBiosciences, San Jose, Calif.).

Tumor Challenge and Immunohistochemistry. Mice reconstituted with BMcells transduced with scTCR-MFG or with a control supernatant werechallenged subcutaneously with various numbers of HPAF tumor cells 5weeks post-reconstitution. Tumor size was measured every 2-3 days usingcalipers. Tumors harvested from control or treated mice were fixed in10% formalin, paraffin embedded, and sections were stained with H&E,anti-Myeloperoxidase (Labvision, Fremont, Calif.), anti-Granzyme B(Labvision), or anti-F4/80 (ebiosciences) in the Department of Pathologycore facility, University of Pittsburgh.

Fluorescent Microscopic Analyses of Tissue Sections From ReconstitutedMice. Six weeks post-reconstitution, C57BL/6 mice or MUC1 Tg. micereconstituted with scTCREGFP transduced BM cells were sacrificed andspleen, lung, and pancreas were harvested and fixed in 2% PFA in PBS.Tissues were then frozen, sectioned and visualized for infiltration withEGFP+ immune cells at the Center for Biologic Imaging, University ofPittsburgh.

Statistics. Statistical analysis was done using Microsoft Excel andGraphpad Prism (GraphPad Software, San Diego, Calif.) software.

In other experiments, we used live imaging microscopy to show thespecificity of the MA TCR for MUC1 by the ability of transfected cellsto flux calcium following stimulation with MUC1+ tumor. One data setshows that control-transfected JRT3-T3.5 didn't flux calcium in responseto stimulation with MUC1+ tumor line (HPAF). A second data set showsthat JRT3-T3.5 cells transfected with the TCR α and β chain from theMUC1-specific CTL clone (MA) fluxed calcium in response to stimulationwith HPAF tumor line. FIG. 8 shows the detection of the scTCR mRNA intransduced BM cells as well as in splenocytes from SCID micereconstituted with BM cells transduced with scTCR-MFG retroviral vectorweeks post-reconstitution. FIG. 9 shows long-term expression of thescTCR on granulocytes in reconstituted C57BL/6 mice 7 monthspost-reconstitution.

The following section describes the RESULTS obtained in this example.

Computational Modeling of MA TCR Binding to the MUC1 “ImmunodominantKnob” Shows Similarity to the Binding of SM3 Antibody Specific for theSame Epitope. MUC1 specific MHC unrestricted CTL clone MA establishedfrom a tumor-draining lymph node of a breast cancer patient waspreviously described (Magarian-Blander et al., J. Immunol. 160:3111-3120 (1998)). This clone mediated TCR-dependent killing of MUC1+tumor cells that was not restricted by their HLA type. It was alsocapable of binding synthetic, tandemly repeated MUC1 epitopesimmobilized on the surface of Polylactide-L-Glycolide (PLGA) beads,resulting in the influx of Ca²⁺. Semi-quantitative RT-PCR analysis andDNA sequencing revealed that the TCR responsible for this binding wascomposed of Vα23.1Jα14.3 and Vβ8.3DβJβ1.2. We cloned this TCR andtransduced the TCR-deficient Jurkat cell line (J.RT3-T3.5) (Ohashi etal., Proc Natl Acad Sci. (USA) 89: 11332-11336 (1992)) with a plasmidvector encoding the full length TCR α and β chains. Its functionalityand specificity were confirmed by live imaging microscopy, whereTCR-transfected cells, but not control cells, fluxed Ca²⁺ upon bindingto MUC1⁺ human tumor cells.

The antibody SM3 recognizes the same epitope, and blocks tumor cellrecognition and killing by MHC-unrestricted MUC1− specific CTL.Considering the common epitope recognized by both SM3 and MA TCR, theircommon Ig-like fold, and similarities in the CDR3 sequences (FIG. 1 a),we used the available sequence and structural information to modelantigen binding by these two functionally related receptors. Both SM3and the MA TCR were modeled by homology using appropriate antibodytemplates. Simulated docking of the MUC1 epitope produced minimizedstructures of both SM3 and MA TCR with bound ligand and predictedsimilar molecular determinants in the CDR2 and CDR3 regions importantfor binding. Our models predict that similarly positioned arginines inthe corresponding Vβ and V_(H) CDR2s interact favorably with theaspartic acid in the MUC1 epitope PDTRP (FIGS. 1 b-1 e). Equivalentlypositioned tyrosines in the corresponding Vα and V_(L) CDR3s stabilizethe interaction with the aspartic acid of MUC1. A glutamine in the αCDR2and a glutamic acid in the βCDR3 interact with the arginine in theepitope, as do an asparagine in the VL CDR2 and a glutamine in the VHCDR3.

MA TCR as a Single-Chain (scTCR) Construct Fused with Zeta (ζ) Chain isFunctionally Expressed on the Surface of T and Non-T Cells. To create amore practical reagent for future gene therapy/immunotherapyapplications, we converted the full length two-chain TCR, which isdependent on CD3 molecules unique to T cells for cell surfaceexpression, into a CD3 independent single-chain TCR (scTCR). The VαJαsegment was fused to the VβDβJβ segment using a 15 amino acid flexiblelinker. This chimeric structure was then cloned into a vector containingthe human TCR Cβ2 and the CD3 ζ transmembrane and cytoplasmic domainsseparated by a short linker, and further subcloned into the pEF6 plasmidto create scTCR-PEF6 mammalian expression vector (FIG. 2 a).Transfection of the scTCR-PEF6 into a CD3⁻ rat basophil cell line RBL(FIG. 2 b) or a CD3⁺ mouse T cell lymphoma line BWZ (FIG. 2 c) resultedin high surface expression of the scTCR, in both cell types.

These same two cell lines were used to test the function and antigenspecificity of the scTCR. Transfected RBL degranulated uponcross-linking of their scTCR with the anti-TCR antibody βF1 or uponspecific recognition of the MUC1 antigen (FIG. 2 d). No degranulationwas seen when cells were stimulated with control antigen ovalbumin.There was no degranulation in control untransfected RBL upon encounterwith either MUC1 or βF1 antibody. Similarly, transfected BWZ cellsproduced a substantial amount of IL-2 when their scTCR was cross-linkedwith plate-bound βF1 antibody (FIG. 2 e). No significant IL-2 productionwas detected when BWZ-scTCR cells were incubated with the DM6 tumor cellline that did not express MUC1; however, substantial level of IL-2 wasproduced upon encounter with MUC1⁺ tumor cell lines HPAF and T3M4. Thesetwo cell lines do not share HLA alleles confirming that cells expressingthe cloned scTCR exhibit the same MHC-unrestricted recognition of MUC1as the original T cell clone from which the TCR was derived. Thedifference in IL-2 secretion in response to these two tumors can beattributed to frequently observed differences in the level of expressionand the extent of glycosylation of MUC1 on different tumor cell lines.

Transduction of Bone Marrow (BM) cells that Differentiate In Vivo intoscTCR⁺ Cells of Multiple Hematopoietic Lineages. In order to test theanti-tumor activity of this MUC1-specific TCR in vivo, we cloned thescTCR into the MFG retroviral vector with an EGFP gene downstream of anIRES sequence (FIG. 3 a). We used the green fluorescence of EGFP totrack scTCR transduced cells. BM cells were isolated from 5-FU treatedBalb/c mice and transduced with the scTCR-EGFP MFG retroviral vectorusing fibronectin-assisted transduction protocol. Seventy-two percent ofBM cells were successfully transduced (FIG. 3 b). We were especiallyinterested in our ability to transduce hematopoietic stem cellscontained within the population of cells that are Thy1.1⁻Lin⁻ c-Kit⁺Sca-1⁺. Thirty-eight percent (38%) of cells of that phenotype weresuccessfully transduced (FIG. 3 e).

Sub-lethally irradiated mice received 2×10⁶ BM cells via tail veininjection. At 3, 6, and 11 weeks post-injection, mice were bled and thepercentages of EGFP⁺ cells in different cell lineages were evaluated. Atweek 3 post-reconstitution, 5.8% of granulocytes (GR-1⁺) were positivefor EGFP (FIG. 4 a). This number increased to 16.3% at week 11.Reconstitution of the monocyte/macrophage (FIG. 4 b) and NK cell (FIG. 4c) lineage followed similar kinetics. Transduced T cells (CD3⁺) wereseen in the periphery at week 6 post-reconstitution and accounted for5.4% (FIG. 4 d) of all T cells. This number dropped to 3.4% at week 11.Similarly, at 6 weeks, 3.4% of B cells (B220⁺) expressed EGFP (FIG. 4 e)and this number dropped to 1.9% at 11 weeks. This is consistent withpublished reports that lymphoid cells transduced with retroviral vectorshave the tendency to silence expression of genes driven by the LTRpromoter.

SCID Mice Reconstituted with the scTCR Transduced BM ControlledOutgrowth of a MUC1⁺ Human Tumor. Based on our findings that the highestpercent of cells expressing the scTCR were those of the innate immunesystem, and that this expression was most stable, we tested thepotential of these cells alone to exert an anti-tumor effect. Wereconstituted lethally irradiated SCID mice with 2×10⁶ BM cellstransduced with the scTCR-MFG retroviral vector or mock-transduced.Expression of the scTCR was detected by RT-PCR in transduced BM cellsprior to injection and in splenocytes from reconstituted mice 60 dayslater (Figure). Mice reconstituted with BM cells transduced with thescTCR retroviral vector or with control supernatant were challenged onemonth later with subcutaneous injection of MUC1⁺ human tumor cell lineHPAF. Mice that received scTCR-MFG transduced BM cells were able toinhibit growth of HPAF tumor cells compared to control mice (FIG. 5 a).The difference in tumor size between the two groups was statisticallysignificant at each time point (p<0.01).

Tumor sections from control mice were intact and homogeneous inappearance, without any infiltration by immune cells (FIG. 5 b, leftpanel). In contrast, tumor sections from scTCR BM reconstituted micewere almost completely destroyed and infiltrated with various immunecells (FIG. 5 b, right panel). The predominant cells in the infiltratewere neutrophils (FIG. 5 c, left) followed by macrophages (FIG. 5 c,mid) and to a lesser extent NK cells (FIG. 5 c, right).

Lack of Autoimmunity in MUC1 Tg. Mice Reconstituted withscTCR-Transduced BM Cells. In order to test whether T cells expressingthis TCR can develop normally in the presence of MUC1, we reconstitutedC57BL6 wild type mice and mice transgenic for human MUC1 (MUC1 Tg.) withscTCR-transduced BM and six weeks later compared the percentages ofscTCR-expressing cells. Table 1 shows that similar percentages of scTCR+T cells as well as other immune cells were seen in C57BL/6 and MUC1 Tg.mice, showing that that there was no selection against thescTCR-expressing cells in MUC1 Tg. mice. Successful reconstitution ofMUC1 Tg. mice allowed us also to determine if the expression of thisreceptor could have deleterious effects on normal tissues expressingMUC1, such as the lung and the pancreas.

TABLE I scTCR-expressing cells in reconstituted C57BL/6 and MUC1 Tg.mice TCR Reconstituted Cell Type (Surface Marker) Control C57BL/6 MUC1Tg. T cells (CD3⁺) 0.2 ± 0^(C)  6.4 ± 1.3  9.0 ± 5.7 B cells (B220⁺) 0.2± 0  9.7 ± 0.3  9.5 ± 1.6 Granulocytes (GR-1⁺) 0.2 ± 0.7 30.8 ± 21.527.4 ± 9.2 NK cells (DX5⁺) 0.1 ± 0 28.5 ± 4.3 18.5 ± 3.7 Monocytes(F4/80⁺) 0.1 ± 0.2 20.4 ± 5.0 14.6 ± 2.7

The analysis was in Table 1 was performed six weeks post reconstitutionwith scTCR transduced BM. C57BL/6 refers to untreated mice. Thenumerical values in Table 1 refer to the mean %±the standard deviation.FIG. 6A shows very few EGFP+ cells infiltrating these tissue and nodifference between wt and MUC1 Tg. mice. There was also no evidence ofdestruction of MUC1 expressing tissues in MUC1 Tg. Mice (FIG. 6B).

One potential explanation for the lack of autoimmunity in reconstitutedMUC1 Tg mice could be that these cells are rendered tolerant or anergicin the presence of MUC1 as a self-antigen. This was not the case,however, since these mice successfully controlled the growth of a mousetumor RMA transfected with human MUC1 (RMA-MUC) much better than thecontrol mice. There was no inhibition of growth of the untransfected RMAcontrol (FIG. 7).

This example shows that a suitable receptor of the invention (such as anMHC-unrestricted TCR specific for an epitope on the tumor antigen MUC1)expressed on cells of the innate and the adaptive immune system is ableto direct the effector functions of these cells specifically againstcancer cells displaying the MUC1 tumor antigen. The MHC-unrestrictednature of the TCR, combined with the stable expression of the MUC1 tumorantigen on a large number of human tumors (over 80%), makes therapybased on this TCR (scTCR) applicable to a large number of patients witha variety of tumors. Our experiments in SCID mice showed that the cellsof the innate system alone can control tumor growth when provided withthe tumor antigen-specific TCR. ScTCR expressing cells were seen asearly as 3 weeks post-reconstitution and were still present at highnumbers more than 7 months later. Both, the early presence oftumor-specific cells and their permanence would be expected to provide abeneficial anti-tumor effect. Furthermore, the persistence of scTCR+cells (FIG. 9) suggests that among the many different cells that weretransduced in the BM, there were also the long-term reconstitutinghematopoietic stem cells that can continue to provide scTCR+ progenitorsand mature cells throughout the life of the animal. Advantageously,expression of this TCR on large percentages of immune and otherhematopoietic cells is not detrimental to the well-being of the animal.Long-term follow up (more than 12 months) of reconstituted micetransgenic for human MUC1 showed no signs of autoimmunity and nospecific infiltration of cells into normal tissues that express MUC1. Wehoped this would be the result because the epitope recognized by thesTCR has been shown to be expressed on the hypoglycosylated MUC1 madeprimarily by tumor cells.

The studies we describe here provide a reasonable expectation that thisreagent is useful for immunotherapy of cancer. For example, a patientcan be treated by transducing this scTCR into bone marrow or peripheralstem cells before infusing the cells into patients who have undergonehigh-dose chemotherapy (HDCT). HDCT followed by autologous stem cell orBM transplant, primarily performed in breast cancer patients, has had alimited, but positive, therapeutic success. The high rate ofpost-transplantation relapse in these patients could be a result of thesurvival of some tumor cells following HDCT treatment or could resultfrom infusing contaminating tumor cells with the stem cell preparation.Vaccination trials aimed at augmenting the immune responses to eradicateresidual tumor cells following stem cell therapy has shown minimalsuccess, probably as a result of the poor and slow reconstitution of theT cell compartment in these patients. The recovery of the innatecompartment of the immune system (in particular NK cells), however,occurs very rapidly after transplantation, reaching normal levels withina month post-transplantation. Accordingly, transducing BM cells with aMUC1-specific TCR or other receptor of the invention prior totransplantation will result in the expression of a tumor-specificreceptor on a high percentage of quickly reconstituting cells of theinnate immune system, which could promptly target and destroy residualtumor cells. Because MUC1 is expressed as a tumor antigen on greaterthan 80% of all human tumors, and the inventive receptor can directeffector cells to all such tumors in virtually all patients, there arefew limits for its clinical application. By selecting a suitableexpression vector, one can target various effector cells for in vitro orin vivo transduction and tailor this type of gene therapy oraimmunotherapy to specific stages of disease and combinations with othertherapies.

The following references can be consulted to better understand thepresent invention and the foregoing example.

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EXAMPLE 2

This example describes the design and construction of several genetransfer vectors for expression in mammalian cells of membrane bound andsoluble human T cell receptors (TCR). In particular, this exampledescribes a vector (TCR α-IRES-β pEF4) that encodes high-levelexpression of a full-length TCR of the present invention on the surfaceof T cells. Furthermore, this example describes a chimeric TCR that doesnot require the presence of endogenous CD3 molecules for surfaceexpression, which allows the receptor to be expressed on cells otherthan T cells. This example also describes a vector encoding a singlechain TCR (scTCR) as a fusion protein of VαVβCβ with CD3ζ.Advantageously, this scTCR is well suited for gene therapy because it isencoded and expressed as a single molecule and does not requireindividual cells to be transduced by multiple nucleic acids. Moreover,this example describes a mammalian expression vector encoding a solublehuman TCR. The approaches used in this example for manipulation of ahuman tumor specific TCR also can be used to study various aspects ofTCR-based immunotherapy.

The following section of this example sets forth the MATERIALS ANDMETHODS used herein.

Primers. Sequences of the oligonucleotide primers (i.e., P1-P15) usedfor cloning are listed in Table 2, along with restriction enzymecleavage and GenBank accession number.

TABLE 2 Primer No. Accession No. Sequence P1 DQ2692125′-CGGGATCCTCGAGATGGAGACCCTCTTGGGCCTGCTTA-3′ (SEQ ID NO: 8) P2 DQ2692135′-CGGGATCCGTCGACATGGCCACCAGGCTCCTCTGCTG-3′ (SEQ ID NO: 9) P3 DQ2692125′-CGGGATCCGGAATTCTCAGCTGGACCACAGCCGCAGCGT-3′ (SEQ ID NO: 10) P4DQ269213 5′-ATAGTTTAGCGGCCGCGGATCCTCAGAAATCCTTTCTCTTGACCA-3′ (SEQ ID NO:11) P5 J04132 5′-GGGGATCCCAAACTCTGCTACCTGCTGG-3′ (SEQ ID NO: 12) P6J04132 5′-TCCCCGCGGCGGCCGCGAATTCTTAGCGAGGGGGCAGGGCCTGCATG-3′ (SEQ ID NO:13) P7 DQ269212 5′-CGGGATCCAGATCCCCACAGGAACTTTCTGGGCTGGGGAAG-3′ (SEQ IDNO: 14) P8 DQ269213 5′-CGGGATCCAGATCCCCACAGTCTGCTCTACCCCAGGCCTCG-3′ (SEQID NO: 15) P9 DQ269213 5′-AGGCGCGCCCCCAGGCCTCGGCGCTGACGATC-3′ (SEQ IDNO: 16) P10 BT019811 5′-AGGCGCGCCGACATGGCCCTGATTGTGCTGGGGGGC-3′ (SEQ IDNO: 17) P11 BT019811 5′-AGGCGCGCCGACGCTGGGGATATGGCCCTGATTGTGCTGGG-3′(SEQ ID NO: 18) P12 DQ2692135′-CTAAGCGTAGTCTGGGACGTCGTATGGGTACAGATCCTCTTCTGAGATGAGTTTTTGTTCTACAACGGTTAACCTGGTC-3′ (SEQ ID NO: 19) P13 DQ2692135′-GCTGCAGGTCAATGGTGATGGTGATGATGCTTGTCATCGTCATCCTTGTAGTCAGCGTCTGCTCTACCCCAGG-3′ (SEQ ID NO: 20) P14 DQ2692125′-ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCCAGGAGGTGACGCAGATTC-3′ (SEQ ID NO: 21) P15 DQ2692125′-CCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGCGGCCCAGGAGGTGACGCAGATTC-3′ (SEQ ID NO: 22)

Cloning of Full-Length TCR α and β Chains. MA CTL clone, the source ofthe TCR, is described in Magarian-Blander et al. (J. Immunol, 160:3111-3120 (1998)). RT-PCR was performed using GeneAmp RT-PCR kit(Applied Biosystems, Foster City, Calif. USA) and using either Vα (P1)or Vβ (P2) leader sequence specific forward primers and Cα (P3) or Cβ(P4) reverse primers. The TCR α and β chains were cloned into themultiple cloning site A (MCSA) and multiple cloning site B (MCSB) in thepIRES vector (Clontech Laboratories, Palo Alto, Calif., USA). TheTCRα-IRES-TCRβ cassette was then subcloned into the pEF4 mammalianexpression vector (Invitrogen, FIG. 10A).

Construction of a Two Chain TCR (tcTCR) and a Single Chain TCR (scTCR)Expression Vectors for Expression on T Cells and Non-T Cells. Human CD3ζ chain was cloned using forward primer (P5) and reverse primer (P6).This cloning strategy maintained an endogenous BamH I site at nucleotidenumber 80 in the extracellular domain of human CD3 ζ. The PCR productwas cloned into the pCDNA3.1 TA vector (Invitrogen). The extracellulardomains of the TCR α and β chains were cloned using (P1) or (P2) forwardprimers and (P7) or (P8) reverse primers. The CD3 ζ/pCDNA3.1 vector wasdigested with BamH I restriction enzyme (New England BioLabs, Beverly,Mass., USA) and the TCR a or chains were cloned in-frame with the CD3 ζchain at the BamH I site (FIG. 11A and FIG. 11B). The TCR αζ and βζ weresubcloned into the pIRES vector and finally the αζ-IRES-βζ cassette wassubcloned into the pLNCX2 (Clontech) expression vector (FIG. 11C). ThescTCR was constructed by cloning the TCR VαJα and joining into the TCRVβDβJβCβ region using flexible linker (GGGGS)₃. The TCR VαJα-VβDβJβCβwas then ligated in-frame to the murine CD3 ζ chain. A linker encoding athrombin cleavage site, GDLVPRGSSRLD (SEQ ID NO: 7), was introducedbetween the TCR β constant and the CD3 ζ transmembrane domain. The scTCRwas cloned into the pEF6 vector.

Construction of scTCR-CD4TM-hζ Mammalian Expression Vectors. The scTCRVαJα-VβDβJβCβ extracellular domain was amplified using Vα forward (P1)and Cβ reverse (P9) and cloned into the pEF6 vector. An Asc I site wasintroduced at the C-terminus in the TCR Cβ region. The human CD4transmembrane (TM) domain fused to the human CD3 ζ cytoplasmic domainwas amplified from the hCD4ζ vector (obtained from Dr. Margo R. Roberts,University of Virginia, VA, USA) using forward (P10) and reverse (P6)primers. Asc I and Sac II sites were introduced into the forward andreverse primers, respectively that allowed in-frame ligation to thescTCR extracellular domain. A modified version of this vector wascreated by inserting a three amino acid (AGD) linker between the TCR Cβregion and the CD4 TM domain. In the latter, PCR was done using (P6) and(P11) primers.

Surface Biotin Labeling and Thrombin Cleavage of scTCR. Surface biotinlabeling and thrombin cleavage was performed using convention methods asdescribed in Engel et al. (Science 256: 1318-1321 (1992)).

Construction of Secreted scTCRs. A soluble, single chain fractionvariable domain (sscFV) encoding the TCR VαJαVβDβJβ or soluble scTCR(sscTCR) domain consisting of the TCR VαJαVβDβJβCβ was cloned intopCDNA3.1 vector by RT-PCR using (P1) and (P12) or (P2) and (P13)primers, respectively. Modified versions of the sscTCR vector werecreated by fusing the sscTCR to the GM-CSF, PCR was done using P14 andP13, or Ig-κ light chain leader sequences (PCR was done using P15 andP13).

Cell Transfection. Human embryonic kidney cells, HEK 293H, weretransfected using lipofectamine 2000 (Invitrogen) according tomanufacturer's instruction. Cells were analyzed for protein expression48-72 hours post-transfection. Jurkat cells were electroporated using aBioRad Gene Pulser II (Bio-Rad Laboratories, Hercules, Calif., USA) at960° F. and 200 V settings.

Stimulation with Superantigen. BWZ and BWZ-scTCR cells were stimulatedwith SEE (Toxin Technology, Sarasota, Fla.). Thirty-six hours later,IL-2 in culture supernatant was measured using murine IL-2 ELISA kit (BDPharmingen, San Diego, Calif.) according to manufacturer'srecommendations.

Purification of Soluble scTCR and Western Blotting. Transfected 293Hcells were grown in DMEM-10. Seventy-two hours after replacing withfresh medium, culture medium was harvested and used for proteinpurification. Anti-HA, c-Myc, and 6-His antibodies were purchased fromSanta Cruz Biotechnology, Santa Cruz, Calif., USA. Anti-Flag M2 antibodywas purchased from Sigma. For constructs encoding c-Myc or HA taggedproteins, sscTCR was purified using Protein G Sepharose beads (AmershamBiosciences) coated with the appropriate anti-tag antibody. For vectorsencoding 6-His tagged proteins, sscTCR was purified using Nickel-Agarosecolumn (Qiagen) according to manufacturer's recommendations.

The following section sets forth the RESULTS obtained in this example.

Reconstitution of the TCR/CD3 Complex on the Surface of JRT3-T3.5 JurkatLine Transfected with the TCR α and β Chain Construct. J.RT3-T3.5 cellslack the TCR β transcript and have low levels of the TCR a chainmessage, and are therefore useful host cells for testing the expressionof transfected TCR. The pEF4 mammalian expression vector was chosenbecause expression of cloned genes is driven by the human elongationfactor-1 alpha (EF-1α) promoter, which is expected to be moretranscriptionally active and stable in T cells than viral promoters. Inaddition, the presence of the IRES sequence permits expression of theTCR α and β chain genes from the same message. This is expected toresult in similar levels of expression of both genes, in contrast togenes driven by different promoters. As shown in FIG. 10, untransfectedJRT3-T3.5 did not express the TCR/CD3 complex on their surface (FIG.10A). Transfection of the TCR β chain alone didn't reconstitute theTCR/CD3 complex on cell surface (FIG. 10B); however, stable transfectionof the TCRα-IRES-β pEF4 vector into J.RT3-T3.5 cells resulted in highlevels of TCR/CD3 complex surface expression (FIG. 10C). Cellstransfected with the TR-ALPHA-IRES-TR-BETA pEF4 vector recognized MUC1+tumors in vitro.

Engineered MUC1-specific Two Chain TCRs (tcTCR) allows Expression on theSurface of Non-T Cells. The requirement for CD3 molecules for expressionof the TCR on the cell surface limits its expression to T cells.Engineered vectors that would bypass this requirement were constructedand allow expression of TCRs and TCR like receptors on other cell types(Engel et al., Science 256: 1318-1321 (1992)). In T cells, theengineered vector eliminates the possibility that the transferred TCRscould pair with endogenous TCR α or β chains that could reduce the levelof the specific receptor on the surface as well as form a potentiallyautoreactive TCR. Construction of the TCR αζ and βζ was done as detailedin the Materials and Methods. As shown in FIG. 11, transfection of theTCR αζ(A) and TCR βζ(B) constructs into human epithelial kidney HEK 293H(non-T cell) cells resulted in surface expression of the TCR α/βheterodimer (D and E). Similar levels of surface expression were alsoobtained when 293H cells were transfected with the TCR αζ-IRES-βζ pLNCX2vector (FIGS. 11C and 11F).

Different Configurations of Single Chain TCRs (scTCRs) were Expressed atVarious Levels on the Surface of 293H Cells. For clinical application,transfection with multiple plasmids can be disadvantageous. Single chain(sc)TCR and single chain antibodies obviate this hurdle and are known inthe art. We made three different MUC1 tumor antigen specific scTCRconstructs and compared the levels of their surface expression (FIG.12). FIG. 12 depicts the level of surface expression of scTCR (FIG.12B), scTCR-CD4TM-hζ (FIG. 12C) and the scTCR-AGD-CD4TM-hζ (FIG. 12D).These scTCRs consist of the TCR antigen binding domain and a signalingcomponent from the CD3 ζ chain. As shown in FIG. 3B, transfection of thescTCR into 293H cells resulted in a substantial increase in surfaceexpression compared to control (A), and transfection with thescTCR-CD4TM-hζ construct gave a much lower level of expression (FIG.3C). However, inserting a 3 amino acids (AGD) linker between the TCR Cβchain and the CD4 TM domain restored high level of surface expression ofthe scTCR (FIG. 3D). While not desiring to be bound by any particulartheory, it is possible that these 3 amino acids provided enoughflexibility to the scTCR to allow proper folding of the molecule andnormal level of surface expression. FIG. 12E shows the same results in amore quantitative way. High surface expression of the scTCR was detectedon both T cells (BWZ murine thymoma) and non-T cells (RBL rat basophilicleukemia) transfected with TCR-pEF6 vector (FIG. 13A). The TCR isfunctional as shown by the ability of the transfected BWZ cells to bestimulated with plate-bound anti-TCR βF1 antibody or with SEEsuperantigen, which binds specifically to the human TCR Vβ8 region (FIG.13B). These cells also recognized MUC1+ tumor cells in vitro (seeExample 1).

Multiple Expression Vectors for Soluble scTCR Production. Soluble singlechain T cell receptors (sscTCRs) can be used as vehicles to deliver andtarget therapeutic drugs to the site of their specific antigen (e.g., atumor expressing MUC1 tumor antigen). Additionally, sscTCR can be usedto study the affinity of interaction between the TCR and its ligandusing, e.g., by Biacore analysis. We successfully generated sscTCR byinserting a thrombin cleavage site between the scTCR Cβ region and theCD3 β transmembrane domain (FIG. 14A). FIG. 14B shows that the scTCRcontaining the thrombin cleavage site can be cleaved from the surface oftransfected cells. Following thrombin cleavage, the scTCR could bepurified from the soluble fraction (FIG. 14C, lane 4) using an affinitycolumn. As expected, the sscTCR has a lower molecular weight than themembrane bound scTCR (FIG. 14C, lanes 2 and 4). The sscTCR can be elutedfrom the affinity column under high pH elution conditions (FIG. 14C,lane 8). Even though this approach was successful in generating sscTCR,the amount that was obtained was extremely low. Other groups havereported expression of a soluble, single chain fraction variable (sscFV)domain of both antibody and T cell receptor. FIG. 15 a shows the designof the sscFV construct that encodes the TCR VαJα-VβDβJβ. FIG. 15 b showsthe same sscTCR construct that was terminated just before the lastcystine in the TCR Cβ region. Two other constructs were designed asdescribed in FIG. 15 b, with the exception of replacing the Vα leadersequence in the sscTCR with either a GM-CSF (FIG. 15 c) or Ig-k lightchain (FIG. 15 d) signal peptide. Various epitope tags were inserted atthe C terminus to facilitate protein expression and purification. Whenthe sscFV construct was transfected into 293H cells, no recombinantsscFV protein could be detected in the culture supernatant (FIG. 15 e,lane a′). However, transfection of the sscTCR construct into 293H cellsresulted in significant amounts of recombinant protein secreted inculture supernatants (FIG. 15 e, lane b′). Transfection of the sscTCRthat was fused to the Ig-κ light chain leader sequence gave a lowerlevel of protein expression than was seen in b′ (FIG. 15 e, lane d′),and transfection of the sscTCR construct fused to the GM-CSF leadersequence yielded no protein secretion (FIG. 15 e, lane c′). Theseresults showed that the presence of the TCR β chain constant region isabsolutely required for expression of the sscTCR. While not desiring tobe bound by any particular theory, we hypothesized that the TCR β chainconstant region must be important for the proper folding of the proteinor that it interacts with and masks other hydrophobic amino acidresidues in the TCR β chain VDJ region, otherwise the scFV is renderedinsoluble. As shown in FIG. 15 f, the recombinant sscTCR could bepurified from culture supernatant using a nickel column. FIG. 15 g showsthe western blot analysis of purified fractions obtained in FIG. 15 f,using anti-FLAG M2 antibody. The expression of soluble form scTCR inthese mammalian cells appears to be sufficiently robust to produce thisreagent for therapeutic purposes or for biophysical analyses.

Thus this example describes several mammalian expression vectors usefulfor functional high-level expression of human TCR α and β chains thatare useful for biological and biochemical analyses, as well asimmunotherapy. Our TCR α-IRES-β pEF4 vector encoding the tumorantigen-specific TCR generated high levels and stable expression of theTCR αβ/CD3 complex on the surface of transfected T cells. We alsoconstructed chimeric TCR αζ and TCR βζ that were successfully expressedon the surface of 293H cells (a non T cell line that doesn't express theCD3 complex). Additionally, we showed that surface expression of the TCRwas dependent on the co-expression of the TCR αζ and TCR βζ. Wehypothesized that pairing of the TCR αζ to the TCR βζ was crucial forproper folding and transport of the heterodimer through the endoplasmicreticulum (ER) and Golgi and eventually to the cell surface.

In contrast to the expression of two chain TCRs, functional scTCRs canbe expressed on the cell surface from a single mRNA transcript. In theexample shown here we constructed a single chain TCR specific for thetumor antigen MUC1 and expressed it on the surface of different celltypes. The expression of the scTCR on the surface of transfected cellswas lower than the level of expression of the native TCR, which weattributed to the presence of charged amino acids in the transmembrane(TM) domain of the CD3 zeta chain that might cause dimerization andretention of the scTCR in the endoplasmic reticulum. In an attempt toincrease the level of surface expression of the scTCR, we replaced theTM domain of CD3ζ in the scTCR with the TM domain of human CD4. However,this new construct was expressed at very low level until we inserted a 3amino acid (AGD) linker between the scTCR constant region and the CD4 TMdomain. High level of surface expression of the scTCR was restored inthis new construct. The short linker provided either flexibility orsufficient spacing between the TCR constant region and CD4 TM to allownormal surface expression.

Most previous attempts to generate soluble TCR were made usingprokaryotic expression systems. However, proteins expressed inprokaryotic cells lack post-translational modifications and may beimproperly folded. In order to avoid these potential problems, we choseto express soluble MUC1-specific scTCR using mammalian expressionsystems. We terminated the scTCR construct just before the last cystinein the TCR□ constant region. Following transfection into 293H cells,large amount of soluble scTCR was detected in culture supernatants.

In conclusion the various constructs we adapted and tested for theexpression of the MUC1-specific TCR can be of interest and help to otherinvestigators interested in TCR immunotherapy or in studying TCR-antigeninteractions.

The following references are useful in understanding the invention, andin particular this example:

-   1. Dudley M E, Wunderlich J R, Robbins P F, Yang J C, Hwu P,    Schwartzentruber D J, Topalian S L, Sherry R, Restifo N P, Hubicki A    M et al: Cancer regression and autoimmunity in patients after clonal    repopulation with antitumor lymphocytes. Science 2002,    298(5594):850-854.-   2. Rubinstein M P, Kadima A N, Salem M L, Nguyen C L, Gillanders W    E, Nishimura M I, Cole D J: Transfer of TCR genes into mature T    cells is accompanied by the maintenance of parental T cell avidity.    J Immunol 2003, 170(3): 1209-1217.-   3. Morgan R A, Dudley M E, Yu Y Y, Zheng Z, Robbins P F, Theoret M    R, Wunderlich J R, Hughes M S, Restifo N P, Rosenberg S A: High    efficiency TCR gene transfer into primary human lymphocytes affords    avid recognition of melanoma tumor antigen glycoprotein 100 and does    not alter the recognition of autologous melanoma antigens. J Immunol    2003, 171(6):3287-3295.-   4. Aarnoudse C A, Kruse M, Konopitzky R, Brouwenstijn N, Schrier P    I: TCR reconstitution in Jurkat reporter cells facilitates the    identification of novel tumor antigens by cDNA expression cloning.    Int J Cancer 2002, 99(1):7-13.-   5. Derby M A, Wang J, Margulies D H, Berzofsky J A: Two    intermediate-avidity cytotoxic T lymphocyte clones with a disparity    between functional avidity and MHC tetramer staining. Int Immunol    2001, 13(6):817-824.-   6. Snyder J T, Alexander-Miller M A, Berzofskyl J A, Belyakov I M:    Molecular mechanisms and biological significance of CTL avidity.    Curr HIV Res 2003, 1(3):287-294.-   7. Weijtens M E, Hart E H, Bolhuis R L: Functional balance between T    cell chimeric receptor density and tumor associated antigen density:    CTL mediated cytolysis and lymphokine production. Gene Ther 2000,    7(1):3542.-   8. Yang S, Linette G P, Longerich S, Haluska F G: Antimelanoma    activity of CTL generated from peripheral blood mononuclear cells    after stimulation with autologous dendritic cells pulsed with    melanoma gp 100 peptide G209-2M is correlated to TCR avidity. J    Immunol 2002, 169(1):531-539.-   9. Magarian-Blander J, Ciborowski P, Hsia S, Watkins S C, Finn O J:    Intercellular and intracellular events following the    MHC-unrestricted TCR recognition of a tumor-specific peptide epitope    on the epithelial antigen MUC1. J Immunol 1998, 160(7):3111-3120.-   10. Engel I, Ottenhoff T H, Klausner R D: High-efficiency expression    and solubilization of functional T cell antigen receptor    heterodimers. Science 1992, 256(5061):1318-1321.-   11. Mittelbrunn M, Yanez-Mo M, Sancho D, Ursa A, Sanchez-Madrid F:    Cutting edge: dynamic redistribution of tetraspanin CD81 at the    central zone of the immune synapse in both T lymphocytes and APC. J    Immunol 2002, 169(12):6691-6695.-   12. Willemsen R A, Weijtens M E, Ronteltap C, Eshhar Z, Gratama J W,    Chames P, Bolhuis R L: Grafting primary human T lymphocytes with    cancer-specific chimeric single chain and two chain TCR. Gene Ther    2000, 7(16):1369-1377.-   13. Novotny J, Ganju R K, Smiley S T, Hussey R E, Luther M A, Recny    M A, Siliciano R F, Reinherz E L: A soluble, single-chain T-cell    receptor fragment endowed with antigen-combining properties. Proc    Natl Acad Sci USA 1991, 88(19):8646-8650.-   14. Eshhar Z, Bach N, Fitzer-Attas C J, Gross G, Lustgarten J, Waks    T, Schindler D G: The T-body approach: potential for cancer    immunotherapy. Springer Semin Immunopathol 1996, 18(2): 199-209.-   15. Gregoire C, Lin S Y, Mazza G, Rebai N, Luescher I F, Malissen B:    Covalent assembly of a soluble T cell receptor-peptide-major    histocompatibility class I complex. Proc Natl Acad Sci USA 1996,    93(14):7184-7189.-   16. Pavlinkova G, Colcher D, Booth B J, Goel A, Batra S K:    Pharmacokinetics and biodistribution of a light-chain-shuffled CC49    single-chain Fv antibody construct. Cancer Immunol Immunother 2000,    49(4-5):267-275.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. An isolated nucleic acid encoding a receptor, other than animmunoglobulin, wherein the receptor binds to a MUC1 tumor antigenindependently of a major histocompatibility complex (MHC).
 2. Theisolated nucleic acid of claim 1, wherein the receptor binds to MUC1tumor antigen with about the same affinity for MUC1 as the MA TCR. 3.The isolated nucleic acid of claim 1, wherein the receptor binds to MUC1tumor antigen with a higher affinity for MUC1 as the MA TCR.
 4. Theisolated nucleic acid of claim 1, wherein the receptor binds to MUC1tumor antigen with a lower affinity for MUC1 as the MA TCR.
 5. Theisolated nucleic acid of claim 1, wherein the receptor is a T cellreceptor. 6-12. (canceled)
 13. The isolated nucleic acid of claim 1,wherein the receptor is a scFv.
 14. The isolated nucleic acid of claim1, wherein the receptor is a T cell receptor (TCR).
 15. The isolatednucleic acid of claim 1, wherein the receptor is a single chainreceptor.
 16. The isolated nucleic acid of claim 1, further comprising alinker of from 1 to about 30 amino acids between the first portion ofthe amino acid sequence and the second portion of the amino acidsequence.
 17. The isolated nucleic acid of claim 1, wherein the receptoris expressed in a T cell better than an otherwise identical receptorlacking the linker in an identical T cell.
 18. The isolated nucleic acidof claim 1, wherein the linker improves the expression of the receptorin a cell in comparison to an otherwise identical receptor lacking thelinker.
 19. The isolated nucleic acid of claim 1, wherein the linkerdoes not have a function selected from the group consisting of animmunological function, a membrane spanning function, a signalingfunction, and a dimerization function.
 20. The isolated nucleic acid ofclaim 1, wherein the Kd of the TCR for a single MUC1 epitope is between0.2 μM and 200 μM.
 21. The isolated nucleic acid of claim 1, wherein thereceptor is soluble.
 22. The isolated nucleic acid of claim
 1. whereinthe receptor is membrane bound.
 23. The isolated nucleic acid of claim1, wherein a cell transduced with the isolated nucleic acid andexpressing the isolate nucleic acid has an avidity (k_(d)) for a cancercell expressing the MUC1 tumor antigen of from about 1×10⁻⁵ M to about1×10⁻¹² M.
 24. The isolated nucleic acid of claim 1, wherein the encodedreceptor does not comprise a constant domain of an antibody.
 25. A celltransduced with the nucleic acid of claim
 1. 26. A lymphocyte transducedwith the nucleic acid of claim
 1. 27. The cell of claim 25, wherein thecell is a T cell.
 28. The cell of claim 25, wherein the cell is a Bcell.
 29. The cell of claim 25, wherein the cell is selected from thegroup consisting of an NK cell, a macrophage, and a dendritic cell. 30.The cell of claim 25, wherein the cell is granulocyte.
 31. A cell otherthan a T cell or B cell comprising a receptor encoded by the nucleicacid of claim
 1. 32. A population of cells isolated from an animalwherein the cells comprise a receptor encoded by the nucleic acid ofclaim
 1. 33. A composition comprising: the nucleic acid of claim 1, anda sterile carrier, pharmaceutically acceptable excipient, adjuvant,and/or buffer that is substantially isotonic.
 34. The composition claim33, wherein the composition is suitable for administration to a mammal.35. The composition of claim 34, wherein the mammal is human.
 36. Acomposition comprising: the cell of claim 25, and a sterile carrier,pharmaceutically acceptable excipient, adjuvant, and/or buffer that issubstantially isotonic.
 37. The composition claim 36, wherein thecomposition is suitable for administration to a mammal.
 38. Thecomposition of claim 38, wherein the mammal is human.
 39. A compositioncomprising: the population of cells of claim 31, and a sterile carrier,pharmaceutically acceptable excipient, adjuvant, and/or buffer that issubstantially isotonic.
 40. The composition claim 39, wherein thecomposition is suitable for administration to a mammal.
 41. Thecomposition of claim 40, wherein the mammal is human.
 42. A genedelivery vector comprising the nucleic acid of claim
 1. 43. The genedelivery vector of claim 42, wherein the gene delivery vector is a viralvector.
 44. The gene delivery vector of claim 42, wherein the viralvector is a retroviral vector.
 45. The gene delivery vector of claim 42,wherein the gene delivery vector is selected from the group consistingof a herpes viral vector, an adenoviral vector, and an adeno-associatedviral vector.
 46. The gene delivery vector of claim 42, wherein the genedelivery vector is a lentiviral vector.
 47. The gene delivery vector ofclaim 42, wherein the gene delivery vector is an “MFG” vector.
 48. Thegene delivery vector of claim 42, wherein the gene delivery vector is anon-viral vector.
 49. The gene delivery vector of claim 42, wherein thegene delivery vector is a liposomal vector.
 50. An isolated orsubstantially purified receptor encoded by the nucleic acid of claim 1.51. The isolated or substantially purified receptor of claim 50, whereinthe receptor is soluble.
 52. The isolated or substantially purifiedreceptor of claim 50, wherein the receptor is membrane bound.
 53. Animmunocytochemistry stain comprising the receptor of claim 50 complexedwith a labeling agent.
 54. An immunocytochemistry stain comprising thereceptor of claim 51 complexed with a labeling agent.
 55. Animmunocytochemistry stain comprising the receptor of claim 52 complexedwith a labeling agent.
 56. (canceled)
 57. A method of activating asignaling pathway in a cell having a signaling pathway, the methodcomprising: a. transducing the cell having a signaling pathway with atleast one nucleic acid encoding a receptor, wherein the receptor i. isexpressed and displayed on the surface of the transduced cell, ii. bindsto a MUC1 tumor antigen independently of an major histocompatibilitycomplex (MHC), and b. contacting the transduced cell to a cellexpressing the MUC1 tumor antigen thereby activating the signalingpathway.
 58. The method of claim 57, wherein the receptor is a T cellreceptor.
 59. A method of activating a signaling pathway in a cellhaving a signaling pathway, the method comprising: a. transducing thecell having a signaling pathway with at least one nucleic acid encodinga receptor, which comprises the nucleic acid of claim 1, wherein thereceptor i. is expressed and displayed on the surface of the transducedcell, ii. binds to a MUC1 tumor antigen independently of an majorhistocompatibility complex (MHC), and b. contacting the transduced cellto a cell expressing the MUC1 tumor antigen thereby activating thesignaling pathway.
 60. The method of claim 59, wherein the receptor is aT cell receptor.
 61. A method of activating a signaling pathway in acell comprising a signaling pathway, the method comprising transducingthe cell with a receptor having affinity for MUC1, wherein the affinityis determined by a first amino acid sequence and a second amino acidsequence, wherein the first amino acid sequence consists essentially ofthe portion of MA Vα23 shown in FIG. 1 (SEQ ID NO:1), and the secondamino acid sequence consists essentially of the portion of MA Vβ8.3shown in FIG. 1 (SEQ ID NO:2).
 62. A method of killing a cancer cell,the method comprising a. isolating a population of cells comprising areceptor, wherein the receptor binds to a MUC1 tumor antigenindependently of an major histocompatibility complex (MHC), and b.contacting the isolated population of cells to a cell expressing theMUC1 tumor antigen thereby killing the cancer cell.
 63. The method ofclaim 60, wherein the population comprises T cells.
 64. The method ofclaim 61, wherein the population consists essentially of T cells. 65.The method of claim 62, wherein the population does not comprise cells,other than T cells, that comprise a receptor that binds to a MUC1 tumorantigen independently of a major histocompatibility complex (MHC).